GSM7115852: Control Pull-Down [GAV012420_004]; Mus musculus; RNA-Seq
PFC was  homogenized and subject to immunoprecipitation-driven RNA extraction according to the TRAP protocol as well as total-RNA extraction for the INPUTs. Briefly, PFC samples were homogenized in the appropriate buffer, centrifuged and supernatant (40 µL) was collected for total-RNA (INPUT), and the remaining supernatant (approximately 400 µL) was transferred to a separate tube and treated with anti-EGFP antibodies and Agarose Plus A/G beads overnight. After centrifugation, the supernatant was removed and the beads, containing ribosome-bound RNA (TRAP samples), were washed and eluted with QIAZOL (Qiagen # 79306). TRAP-RNA and total-RNA samples were extracted using the miRNeasy Mini Kit according to manufacturer's instructions. RNA integrity numbers (RIN)for eachRNA sampleweredetermined using an AgilentTapestationand samples were subjected to rRNA depletion and sequencing. The RNAseq libraries were prepared for Illumina sequencing usinf CORALL Total RNA-seq Library Prep Kit with Unique Dual Indices. Final amplified libraries were purified, quantified and average fragment sizes confirmed to be 254 -323 and then subjected to test sequencing on MiniSeq instrument in order to check sequencing efficiencies. Sequencing was carried out on NovaSeq 6000 (Illumina), SP flowcell, 2x50 bp PE reads.